Our results revealed that majority of iPSC lines, generated both from SBA and healthy donors were pluripotent and able to differentiate toward all three germ layers (Supplementary Fig.?S1A). NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches. Introduction Neural tube defects (NTDs) are a common type of congenital anomaly. The manifestation of NTDs occurs in very early stages of embryonic development, with failed closure of the neural tube at around day 281. An occurrence frequency of the disease is 1 case per 1,000 births2,3 and each year nearly 300,000 infants with NTDs are born, further resulting in death or lifelong disabilities4. Therefore, socioeconomic cost associated with NTD patients is very high due to the increased morbidity and premature mortality5. Spina bifida aperta (SBA) is one of the most severe types of NTDs associated with herniation of neural tissue through an incompletely formed spine. SBA is Zofenopril a progressive, nonlethal but yet chronic disease with significant morbidity6,7. The condition can be easily picked up in first trimester Pdgfra screening programs. However, in practice, most diagnoses are still made in the second trimester8. Although fetal surgery by prenatal repair of the damage is a common treatment approach for SBA8, the malformation may lead to severe progressive complications after birth, like hydrocephalus, cognitive impairments, and sensory-motor deficits6,7. The aetiology of SBA, and NTDs in general, is poorly understood9,10. Preclinical studies performed on mice showed numerous genes associated with the disease; however, specific genes described in mice are not sufficient to explain the heterogeneity of NTDs in humans1,9. Over 25 years of clinical and experimental studies indicate that NTDs arise from a combination of genetic and gene-environment interaction factors. The risk of NTDs is greatly reduced by folic acid (FA) taken as a supplement starting from at least one month before conception and continuing throughout the first trimester of pregnancy1,11. The folate metabolic pathway plays a crucial role in nucleotide biosynthesis, proper cell proliferation and generation of methyl donors12C14. Moreover, during the first-trimester of pregnancy, exposure to FA antagonists is associated with an increased risk of congenital anomalies, including NTDs15. For instance, exposure to the folate antagonist methotrexate (MTX) induces NTDs in animal models16. MTX inhibits dihydrofolate reductase (DHFR) an enzyme that participates in the tetrahydrofolate (THF) synthesis from folate17. However, the exact mechanism through which MTX causes and FA supplementation prevents NTDs remains unknown1. High and persisting proliferation of neural stem cells (NSCs) is required for the normal development and correct morphogenesis of the central nervous system (CNS). FA, influences the proliferation and differentiation of NSCs, whereas MTX impairs cell proliferation of embryonic NSCs in animal models18. Consistent folate deficiency can also lead to various neurological Zofenopril conditions in children and adults19,20. Folate transport through its own receptors might be crucial to prevent NTDs, as, for instance, Folate Receptor 1 (receptors are expressed on the plasmatic membrane of the human placenta23, where they play a role in folate transport during early embryonic development. The mechanistic link between and NTDs is also unclear. Thus, models of human NTDs to capture the pathological phenotype and reveal the mechanism of FA action are urgently needed. Human induced pluripotent stem cell (iPSC) technology could provide an attractive model to recapitulate the disease with its specific mechanisms and address early events in NTD manifestation. During the early neural differentiation, pluripotent stem cells (PSCs) undergo morphogenetic Zofenopril events and form radially arranged columnar epithelial cells, named neural rosettes. Neural rosettes resemble the structure of embryonic neural tube and express several early neural and radial glia (RG) markers, including PAX6, SOX1, NESTIN, BLBP (known also as FABP7) similar to the developing neural tube24C26. Cells within rosettes acquire apical-basal polarity based on the localized expression of adherence and tight junction proteins, including N-CAD and ZO-1 in a ring surrounding the central lumen27,28. The structures, proliferation potential and proper polarization of the neural rosettes are significant features for their neurogenic potential25,27. Thus, generation of the neural rosettes from iPSCs derived from NTD patients and determination of the disease features and mechanisms are of utmost importance to allow intervention strategies to be proposed. Musculoskeletal problems are common among SBA patients and in animal models29,30, confirming the complexity of the disease and its involvement in various tissues. Several genes associated with NTDs, including Pax331,32 in mice, play an important role in mesodermal derivatives and loss of mesoderm in zebrafish leads to NTDs33. Thus, the exploration of the human NTD characteristics in mesodermal derivatives is timely. In the present study, we established a human NTD Zofenopril cell model using disease-specific iPSCs. We described the morphological features of the disease administrated or not with FA during early neural and mesodermal lineages specification. We also.